High-resolution image reconstruction in fluorescence microscopy with patterned excitation

Abstract

We discuss data treatment strategies in structured illumination microscopy, using simulated and experimental data. In the setup, the illumination pattern is generated by projecting a movable pinhole mask into the sample, and a wide-field fluorescence microscope image is acquired for each pattern position. The structured illumination data obtained from a two-dimensional illumination pattern can be treated by projection strategies such as in video confocal microscopy (sum, maximum, maximum minus minimum, and superconfocal), by a scaled subtraction of the out-of-focus estimate, or by a modified version of the Fourier-space treatment, as is known for data from one-dimensional structured illumination. We investigate the influence of some data processing strategies on unwanted effects such as residual patterning and local deviations from linearity in the reconstructed intensity.